As an important building block for the synthesis of angiotensin-converting enzyme inhibitors, ethyl (R)-2-hydroxyl-4-phenylbutanoate [(R)-HPBE] has attracted increasing attention. The key to industrial biosynthesis of (R)-HPBE is a biocatalyst that efficiently reduces ethyl 2-oxo-4-phenylbutanoate (OPBE) with high R enantiose-lectivity. This paper proposed a strategy for identifying key residues involved in enantioselectivity control based on per-residue free energy decomposition and sequence conservatism analysis. Using this strategy, 4 noncon-servative sites with high energy contribution to binding of OPBE were chosen as engineering targets, generating variant Mu27 with 99% conversion and 98% (R) ee value at substrate loading of up to 500 mmol/L. MD simulations suggested the higher stability and formation probability of Mu27-OPBEproR prereaction state as key reasons for the excellent R-enantioselectivity of Mu27 towards OPBE. The success in this study provides a viable approach for rational design of alcohol dehydrogenases with high enantioselectivity towards unnatural substrates.

中文翻译：

---

基于每个残基的自由能分解和序列保守性分析，重新设计用于不对称合成（R）-2-羟基-4-苯基丁酸乙酯的短链脱氢酶/还原酶

作为合成血管紧张素转化酶抑制剂的重要组成部分，（R）-2-羟基-4-苯基丁酸乙酯[[-HPBE]]引起了越来越多的关注。（R）-HPBE工业生物合成的关键是一种生物催化剂，该催化剂可有效还原具有高R对映体选择性的2-氧代-4-苯基丁酸乙酯（OPBE）。本文提出了一种基于残基自由能分解和序列保守性分析的对映选择性控制中关键残基的识别策略。使用该策略，选择了4个对OPBE结合具有高能量贡献的非保守位点作为工程目标，在高达500 mmol / L的底物负载下产生了99％的转化率和98％（R）ee值的变体Mu27。MD模拟表明，Mu27-OPBEproR预反应态具有更高的稳定性和形成可能性，这是Mu27对OPBE优异R对映选择性的关键原因。这项研究的成功为合理设计对非天然底物具有高对映选择性的醇脱氢酶提供了可行的方法。